Extragonadal Actions of FSH: A Critical Need for Novel Genetic Models

Abstract

Follicle-stimulating hormone (FSH) is critical for ovarian folliculogenesis and essential for female fertility. FSH binds to FSH receptors (FSHRs) and regulates estrogen production in ovarian granulosa cells to orchestrate female reproductive physiology. Ovarian senescence that occurs as a function of aging results in loss of estrogen production, and this is believed to be the major reason for bone loss in postmenopausal women. Although conflicting, studies in rodents and humans during the last decade have provided genetic, pharmacological, and physiological evidence that elevated FSH levels that occur in the face of normal or declining estrogen levels directly regulate bone mass and adiposity. Recently, an efficacious blocking polyclonal FSHβ antibody was developed that inhibited ovariectomy-induced bone loss and triggered white-to-brown fat conversion accompanied by mitochondrial biogenesis in mice. Moreover, additional nongonadal targets of FSH action have been identified, and these include the female reproductive tract (endometrium and myometrium), the placenta, hepatocytes, and blood vessels. In this mini-review, I summarize these studies in mice and humans and discuss critical gaps in our knowledge, yet unanswered questions, and the rationale for developing novel genetic models to unambiguously address the extragonadal actions of FSH.

Controversy exists with regard to extra-gonadal actions of FSH. This mini-review summarizes the controversies, identifies the critical gaps, and provides the rationale to develop new genetic models.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by gonadotrope cells in the anterior pituitary (1, 2). In the female, FSH binds to G protein‒coupled FSH receptors (FSHRs) expressed on ovarian granulosa cells and promotes estrogen production (3, 4). Both physiological and genetic studies using rodent models and human patients carrying mutations in the hormone-specific β-subunit‒ and FSHR-encoding genes indicate that FSH action is essential for antral-stage follicle development during ovarian folliculogenesis and, consequently, female fertility (5–8).

The aforementioned classical dogma in female reproductive physiology has been challenged in the last decade with the startling discovery that FSH directly regulates bone mass (9). Shortly after, this was followed by several reports claiming expression of extraovarian FSH receptors on different tissues (10–12), including more recently on adipocytes (13, 14). More recently, a subset of these expression studies was reevaluated by a systematic and detailed expression analysis (15). This group challenged some of the original expression studies, in particular previous reports in which expression of FSHRs on endothelial, uterine, and placental cells and signaling in these cells were reported (10–12). Thus, although the observation that FSH exerts multiple actions in nongonadal tissues is exciting and thought-provoking, controversy clearly exists regarding nongonadal FSHR expression itself, let alone FSH actions in these nonclassical tissues.

In this review, I discuss some critical gaps in our existing knowledge regarding the extragonadal actions of FSH, with a special emphasis on FSH actions on bone, and provide the rationale for developing novel genetic models.

Mouse Models Provide Genetic Evidence for FSH Actions on Bone

A little over a decade ago, Sun et al. (9) reported that FSH directly regulates bone mass. In genetic models missing either the FSH ligand (Fshb null mice) or FSHRs (Fshr null mice) throughout the body from birth, the net bone density was shown to be maintained or increased in a Fshb/Fshr gene dosage‒dependent manner and independent of estrogen status (9). Several observations strongly support these genetic studies. First, in the face of normal/declining estrogen levels, women experiencing perimenopausal transition maximally lose bone density, and this is strongly correlated to high levels of serum FSH (9). Second, a large cohort of European women who were harboring polymorphisms in FSHR that lead to constitutively active FSHRs rapidly lost bone density (16). Third, loss of bone density is prevented in ovariectomized female mice lacking FSHRs (9). Finally, live imaging studies in which near-infrared fluorophore‒coupled recombinant FSH ligand was injected into adult mice identified intense labeling of bones by FSH (17).

Sun et al. (9) further demonstrated two other important aspects of FSH action on bone. First, they used bone marrow precursors, primary mouse/human osteoclasts, osteoclast cell lines, osteoblasts, and osteoblast cell lines and demonstrated the presence of FSHRs exclusively on “bone-chewing” osteoclasts but not on bone-forming osteoblasts. They used a variety of expression methods, including reverse transcription polymerase chain reaction, Western blot, cell isolation by fluorescence-activated cell sorting, and osteoclast cell surface immunolabeling with antibodies against FSHR (9). Furthermore, full-length and truncated FSHR promoter fragments fused to luciferase were active in RAW-C3 osteoclast precursor cells upon receptor activator of nuclear factor κB ligand treatment. Sun et al. (9) demonstrated that FSH did not affect osteoblasts; it stimulated osteoclastogenesis and bone resorption in a series of in vitro studies. That these effects on osteoclasts were specific to FSH was further illustrated by the absence of any effect by luteinizing hormone (LH), a hormone coexpressed with FSH in gonadotropes, and by gonadotropin-releasing hormone (GnRH), the hypothalamic hormone that regulates secretion of both LH and FSH (9).

Second, Sun et al. (9) identified that FSH actions in mouse/human osteoclasts are mediated via G_i2α, unlike those in ovarian granulosa cells in which G_Sα is the main coupling protein that activates the cyclic adenosine monophosphate–dependent pathway. Interestingly, FSH activates Erk, Akt, and IκBα in osteoclast cells, somewhat similar to its regulation of Amh promoter expression in Sertoli cells (18). In osteoclast cells, G_i2α but not G_i1α or G_i3α is indeed the predominant form expressed. FSH downregulates cyclic adenosine monophosphate production, and FSH-induced phosphorylation of Erk, Akt, and JNK proteins could be blocked by specific inhibitors to each of these signaling pathways in osteoclasts. Finally, FSH failed to induce c-fos accumulation into nuclei of mouse osteoclasts lacking the G_i2α protein (9, 18).

Subsequent studies (i.e., to those mentioned previously) identified that FSH regulates FSHR gene transcription in osteoclasts and human monocytes that give rise to osteoclasts (19, 20). However, the FSHR abundance in these cells was much less than that in ovarian cells. Furthermore, truncated FSHR-encoding messenger RNA was found in these cells as a result of alternative splicing in the FSHR gene in exons 8 to 10 (19). These observations are similar to the scenario found in sheep ovarian cells, which express different splice variants, each coupled to a distinct signaling pathway downstream (21, 22). The mechanisms underlying differential G protein coupling by the FSHR are not yet defined. Most important, FSH action in the osteoclast linage cells leads to the production of cytokines, including tumor necrosis factor α (TNF-α), the typical inflammatory cytokine (23). Interestingly, Fshb null mice have lower TNF-α levels, and mice lacking TNF-α are indeed resistant to hypogonadal bone loss in the presence of high FSH levels compared to controls. Thus, it was suggested that TNF-α is critical to the effect of FSH on bone mass (23).

Consequences of Immunoneutralization of FSH Action on Bone and Adipocytes

An important clinical implication of the detrimental effects of elevated FSH levels on bone even in the presence of normal estrogen levels is to prevent bone loss in perimenopausal and postmenopausal women. To this end, a blocking antibody to the FSHβ subunit was developed for future therapeutic advantage in humans and to definitively distinguish direct effects of FSH from the opposing effects of estrogen on bone (24). In this immunoneutralization approach, a goat polyclonal anti-peptide antibody was developed against an FSHR-binding region of mouse FSHβ comprising 13 amino acids in length. In an enzyme-linked immunosorbent assay, this antibody bound both the peptide and full-length FSH‒coated plates, and the immune complexes could get precipitated by an anti-goat immunoglobulin G. When intraperitoneally injected, the blocking antibody significantly reduced ovariectomy-induced bone loss in mice (24). Surprisingly, the blocking FSHβ antibody stimulated bone formation, most likely via blocking FSHR-mediated effects on mesenchymal stem cells (24).

In addition to bone loss, the late perimenopausal transition is associated with enhanced visceral adiposity and is coincident with disrupted energy balance and reduced physical activity. At this and later stages of menopause, the effects of loss of estrogen action on energy balance are less understood. Thus, blocking of FSH action to simultaneously benefit bone loss and adiposity accumulation associated with menopause was tested (14). The previously validated anti-peptide FSH polyclonal antibody was further validated on the basis of human FSHβ-FSHR crystal structure and was found to inhibit FSH binding to its receptor. In addition, a monoclonal antibody raised against the corresponding human peptide (i.e., that differed by two amino acids at positions 10 and 11) also recognized and cross-reacted with the mouse peptide (14).

When injected into wild-type mice on a high-fat diet, the FSH polyclonal antibody caused a reduction in both fat mass measured by quantitative nuclear magnetic resonance analyses and total, visceral, subcutaneous fat volume as measured by micro‒computed tomography (14). Moreover, the antibody reduced adiposity in ovariectomized mice. Consistent with these effects of FSH signaling on adipose tissue, immunostaining with FSHR antibodies revealed intense FSHR staining in white (inguinal and visceral) and brown adipose tissues (14). Strikingly, the blocking antibody activated the mitochondrial uncoupling protein 1, enhanced mitochondrial biogenesis, and triggered white-to-brown adipose tissue conversion. Thus, the FSHβ anti-peptide antibody appears to be a potential dual-purpose reagent that could have promising clinical applications in the future in treating both osteoporosis and obesity in postmenopausal women (14).

A recent study examined the correlation between elevated FSH and serum lipid levels in 400 Chinese postmenopausal women (25). At least twofold-elevated FSH levels correlated to higher serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. In this study, FSHR expression was identified in mouse liver tissue and human liver cancer cells (HepG2) by polymerase chain reaction, Western blotting, and immunolocalization (25). FSH directly inhibited low-density lipoprotein receptor (LDLR) in a dose- and time-dependent manner in HepG2 liver cancer cells. Knockdown of FSHRs reversed the lower LDLR level induced by FSH. This study identified the hepatocyte as another target cell for FSH action. The proposed mechanism involves FSH interaction with its receptors in hepatocytes to reduce LDLR levels, which subsequently blocks the endocytosis of LDL-C and elevates circulating LDL-C levels (25).

Studies That Contradict Direct Actions of FSH on Bone

Although loss-of-function FSH mouse models support the concept that an elevated FSH level is detrimental to bone, ectopically expressed human FSH in a gain-of-function transgenic mouse model contradicted this notion (26). In this model by Allan et al. (27), human FSH subunit‒encoding transgenes were first targeted predominantly to liver. Later, they were introduced onto a hypogonadal (hpg) genetic background (as a result of functional GnRH deficiency, this mouse lacks endogenous gonadotropins and steroids), thus creating an LH- and estrogen-deficient environment. Human FSH expression in this model resulted in dose-dependent de novo bone formation and caused an increase in bone mass. These mice also demonstrated increased serum inhibin-A and testosterone levels, and ovariectomy abolished a FSH-dependent increase in bone formation (26). Expression analyses failed to detect FSHRs in mouse bone or cultured bone cells (osteoclasts and osteoblasts). Together, these data indicate that FSH acts in conjunction with an ovarian-dependent mechanism and indirectly regulates bone mass (26). Because of low levels of Fshr expression in bone cells, it is plausible that Allan et al. (26) did not detect FSHR messenger RNA because they did not perform gene-specific reverse transcription, unlike in the studies reported by Robinson et al. (19).

Previous studies attributed some of the obesity and skeletal phenotypes in Fshr null female mice to an estrogen deficiency (28). Another study similarly indicated the predominant role of estrogen deficiency and ovarian products was independent of FSH effects on bone resorption (29). A third study similarly refuted FSH actions on bone in male mice and human osteoclasts in vitro (30).

Clinical studies in humans using GnRH agonists to suppress FSH levels and analyze the consequences provided discordant results compared with what we know about FSH actions on bone and adiposity in mice. Drake et al. (31) reported and Woodruff and Kohsla (32) commented that FSH suppression in postmenopausal women had no effect on bone resorption. FSH suppression concomitant with that of steroids resulted in an increase in fat mass in another study (33). In addition, blockade of androgen action by GnRH agonist treatment in patients with prostate cancer resulted in bone loss and increased fracture risk, confirming that FSH suppression had no beneficial effect in these men (34).

Critical Gaps in Our Knowledge on Extragonadal Actions of FSH

A summary of extragonadal expression of FSHRs and the putative actions of FSH in these tissues/cells is presented in Table 1. Recent studies in rats provided controversial data on FSH regulation of bone mass. Some of these studies using rats (37–39) are in agreement with those by Sun et al. (9), and some studies (40) support the studies by Allan et al. (26). Similarly, a survey of several clinical studies indicated that a clear association exists between FSH and bone resorption. At least three studies indicate that FSH levels predicted bone parameters independently of other hormones or factors (41, 42). On the contrary, several clinical studies did not indicate any correlation between FSH and bone loss (31, 32, 43).

Table 1.

A Summary of Extragonadal Expression of FSHRs and Putative Actions of FSH

Species	Extragonadal Tissue/Cell Tissue Type	FSH Action Noted	Reference
Mouse	Bone (osteoclasts)	Bone resorption	Sun et al. (9)
Human	Umbilical vein endothelial cells, placenta	Angiogenesis
Human and mouse	Female reproductive tract	Myometrial contractility	Stilley et al. (10, 11)
Human	Liver (hepatocytes)	Regulation of LDLR levels	Song et al. (25)
Chicken	Adipose	Stimulation of lipid biosynthesis	Cui et al. (35)
Human and mouse	Adipose	Beiging and mitochondrial biogenesis, activation of brown adipose tissue and enhances thermogenesis	Liu et al. (14, 36)

A systematic examination of all the previously mentioned studies (basic and clinical) exposes several critical gaps in our knowledge of the extragonadal actions of FSH. First, in lieu of the recent study that questioned the extragonadal expression of FSHRs (15), it is important to thoroughly validate the reported commercial antibodies against FSHR in independent laboratories. These antibodies must be tested on Fshr null mouse tissues using immunolocalization and Western blot techniques. Second, no study has tested the direct binding of a ¹²⁵I-radiolabeled recombinant or purified FSH preparation to nongonadal tissues/cells in a competition assay with the corresponding antigen as cold hormone. It may be possible that contaminating proteins in pure FSH preparations or FSH itself can nonspecifically bind to FSHR-like proteins or another distinct binding protein expressed on nongonadal tissues/cells. Third, if the nongonadal FSHR is biochemically distinct (e.g., truncated FSHR variants have been proposed) compared with the full-length bona fide gonadal receptor, then it may be biochemically purified and characterized from these nongonadal tissues (i.e., if the receptor density is sufficient enough to permit direct purification). Alternatively, one could express the cloned truncated FSHR complementary DNA found in nongonadal cells into heterologous cells in high enough numbers to potentially permit biochemical isolation. Fourth, to explain the discordant results among different clinical studies that used a GnRH agonist to suppress FSH levels, the age of agonist-treated women may be critical. This age could be between perimenopausal to early menopausal transition, when bone loss appears to be maximal, FSH levels are elevated, and estrogen persists.

Finally, some unanswered questions remain. The mechanism of stimulated bone formation in mice upon blocking of FSH action with a FSHβ polyclonal antibody remains to be explored (14). It is not entirely clear why FSH actions on bone manifest as a function of aging in women. Serum FSH levels may be examined in women with premature ovarian failure or associated early-onset ovarian defects to determine whether they correlate to bone mass or bone markers in these patients. It is also not clear if sex-specific differences exist regarding FSH actions on bone in men who undergo andropause (44, 45), equivalent to menopause in women. Whether these men have elevated FSH levels and whether suppressing or blocking FSH has any effects on bone loss in these aged men may be determined.

Novel Genetic Models Can Resolve Controversies of Extragonadal FSH Actions

Three new genetic models described next may help resolve the extragonadal actions of FSH in mice. Regardless of whether full-length or truncated FSHRs are expressed on extragonadal mouse tissues, the cell-autonomous role of FSHRs can be directly tested using a tissue/cell-specific deletion of Fshr by a Cre-lox approach. Fshr^flox/+ mice are first generated by either conventional gene targeting in embryonic stem cells or by using the recently developed CRISPR/Cas9–based precise genome-editing methods (46). Next, Fshr^flox/flox mice are derived from these mice and crossed to tissue/cell-specific Cre recombinase‒expressing driver lines. For example, FSHR-mediated signaling in bone can be tested by crossing Fshr^flox/flox mice independently with either osteoblast-specific or osteoclast-specific Cre-expressing lines. This strategy allows testing the loss of FSHR signaling in a given tissue/cell type and is dependent on when Cre expression is initiated during development. A modified version of this strategy allows spatiotemporal deletion of Fshr in desired nongonadal tissues/cells and involves regulating CRE expression by an inducible system at desired times (47).

FSH ligand expression can also be temporally regulated in mice. In a second model, a human FSHB transgene is targeted to pituitary gonadotropes and expressed using a tetracycline-inducible system (48, 49) and on a Fshb null genetic background. This genetic rescue model should permit expression of an interspecies hybrid FSH heterodimer (mouse α, human FSHβ) in the absence of endogenous FSH and at desired times. This system allows expression of high levels of FSH independently of chronological age, and the effects are determined directly on a given extragonadal tissue.

Finally, mice with a temporal Fshb deletion can be developed in a third genetic model. This is in contrast to the existing Fshb null mice in which FSH is absent from birth. Fshb^flox/+ mice are first generated by one of the previously discussed gene manipulation methods from which Fshb^flox/flox mice are then obtained. These floxed alleles are then recombined by “turning on” an inducible Cre expression system and achieving Fshb deletion exclusively in gonadotropes at desired times (47). The consequences of absence of the FSH ligand can then be studied in various nongonadal tissues at a given time point. However, it must be noted that generating these three mouse models is laborious, time-consuming, and expensive.

Conclusions and Final Remarks

The discovery that FSH directly regulates bone mass and adiposity is clearly a paradigm shift in our understanding of mouse pituitary-ovarian-bone physiology. Human studies are currently controversial, and definitive answers are yet to come. If the extragonadal action of FSH is also confirmed in humans, novel therapeutic opportunities for treating postmenopausal osteoporosis and increased adiposity will be feasible. Low-dose estrogen therapy combined with blocking of FSH action may prove highly effective for women who are prone to a high risk of bone fractures and adverse cardiometabolic functions. If carefully conducted studies blocking FSH action at an early perimenopausal age in women fail to confirm the results from mouse studies, it will suggest species-specific differences between mice and humans.

An additional point to consider is the sex bias in most of the published studies examining correlations between elevated FSH levels and bone loss/function in women. An often-ignored aspect of estrogen/FSH actions on bone is the contribution by other gonadal factors, such as inhibins and activins, and these must be assessed in the future.

One of the hallmarks of FSH biosynthesis in the aging pituitary is altered N-glycosylation, a key posttranslational checkpoint event (50, 51). Age-dependent human FSH glycoforms have been biochemically identified, purified recombinant forms expressed, and their bioactivities tested in in vitro and in vivo assays (52–55). Whether these FSH glycosylation variants differentially affect different gonadal and nongonadal target tissues/cells may be important to address in the future. Finally, new genetic models to be developed in the future, previous studies with mice and humans, and ongoing and future clinical studies will resolve the controversies of FSH actions on nongonadal tissues. These studies may also eventually enhance understanding of the evolutionary implications of why an ancient hormone selected for procreation turns out be devastatingly detrimental for bone and adipose tissues, afflicting millions of older women.